There have been known various nucleic acid amplification methods, such as an LAMP (Loop-Mediated Isothermal Amplification) method, an ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, and a PCR (Polymerase Chain Reaction; hereinafter, referred to as “PCR” in some cases) method. Among these, the PCR method has been widely used to amplify a nucleic acid, in the molecular biology field. In recent years, the PCR has been used more widely, so that it is applied not only to the molecular biology field but also to detection of a pathogen, detection of a substance (such as an allergen) mixed in a food product, livestock management, detection of a single nucleotide polymorphism (hereinafter, referred to “SNP” in some cases), etc.
Any usage of the PCR requires detection of a nucleic acid fragment amplified by the PCR. A great number of methods have been already known as a method for detecting an amplified nucleic acid fragment amplified by the PCR.
A most typical method for detecting the amplified nucleic acid fragment is such that (i) a solution which has been subjected to an amplification reaction is applied to agarose gel electrophoresis, and treated with a fluorescent intercalator, such as an ethidium bromide, for binding to the amplified nucleic acid fragment, and (ii) thereby the amplified nucleic acid fragment is observed for specific fluorescence.
Other than this method, for example, Patent Literature 1 discloses a method in which a nucleic acid amplification reaction is carried out with the use of a fluorescently-labeled primer, so that an amplified nucleic acid product is detected by means of fluorescence polarization. Further, Patent Literature 2 discloses a method for detecting an amplified nucleic acid fragment by (i) passing polarized light through a reaction solution subjected to the nucleic acid amplification reaction, and (ii) measuring an angle of rotation or a circular dichroism of the polarized light.
Furthermore, Patent Literature 3 discloses a method for detecting whether or not the nucleic acid amplification occurs, by (i) amplifying a target region on a polynucleotide chain, and (ii) detecting precipitating magnesium pyrophosphate while the amplification reaction proceeds. Moreover, Patent Literature 4 discloses a method in which (i) pyrophoric acid, produced in a polymerase extension reaction based on a specific base sequence of a target nucleic acid fragment, is treated with an enzymatic reaction reagent containing an oxidase, (ii) electron transfer that occurs during the action of the oxidase is amplified under the presence of an electrochemically active intercalator, and (iii) the amplified electron transfer is electrochemically detected as a current.
The techniques disclosed in Patent Literatures 1 through 4 allow the detection of the amplified nucleic acid fragment amplified by the nucleic acid amplification reaction. For the detection of the amplified nucleic acid fragment amplified by the PCR, any nucleic acid detection method is applicable.
As the nucleic acid detection method, for example, Patent Literature 5 discloses a method for detecting a nucleic acid by enzymatically coloring a nucleic acid sequence immobilized to a solid support.
Further, Patent Literature 6 discloses a method for detecting a nucleic acid by dyeing the nucleic acid with a metal and reacting the metal-dyed nucleic acid with a dye precursor and an auxiliary agent.